Optimization of the fluorogen-activating protein tag for quantitative protein trafficking and colocalization studies in S. cerevisiae

Spatial and temporal tracking of fluorescent proteins (FPs) in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active FPs fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations. To help study protein subpopulations, bioconjugate tags, including the fluorogen activation proteins (FAPs), were developed. FAPs are comprised of two components: a single-chain antibody (SCA) fused to the protein of interest and a malachite-green (MG) derivative, which fluoresces only when bound to the SCA. Importantly, the MG derivatives can be either cell-permeant or -impermeant, thus permitting isolated detection of SCA-tagged proteins at the cell surface and facilitating quantitative endocytic measures. To expand FAP use in yeast, we optimized the SCA for yeast expression, created FAP-tagging plasmids, and generated FAP-tagged organelle markers. To demonstrate FAP efficacy, we coupled the SCA to the yeast G-protein coupled receptor Ste3. We measured Ste3 endocytic dynamics in response to pheromone and characterized cis- and trans-acting regulators of Ste3. Our work significantly expands FAP technology for varied applications in S. cerevisiae.


Supplemental Figures & Legends
(A) Cytometry analysis of WT cells either expressing soluble FAPORIGIN or expressing no fluorophore (negative control for non-fluorescent cells).These nonfluorescent cells were used as a control to remove background cell autofluorescence or non-expressing cells (as occurs sometimes in small subpopulations when plasmids are used) by gating.(B) The percentage of cells remaining in each experiment performed for Figure 2D after the non-fluorescent cells were removed by gating, the threshold for which was established in panel S1A.In all cases, ~80% of cells remained and there was no significant difference between FAPORIGIN or FAPOPTIM constructs in the percentage of non-fluorescent cells identified in these assays.Student's t-tests were used to compare FAPOPTIM to FAPORIGIN for each construct (not significant = ns).To amplify the FAP-KANMX4 cassette for C-terminal integration cloning use the F2 (indicated as FOR on map) and R1 (indicated as REV on map) primers, as described in (Longtine et al., 1998), with gene-specific primer extensions added at the 5' end of each primer.The MCK7 coding sequence was replaced with the hygromycin resistance cassette using the (Longtine et al., 1998) strategy in the BY4742 background.yps1∆ mkc7∆

pRS415-TEF1pr-MFA-NFAPoptim
TEF1pr-MFA1 signal sequence-NFAPoptim CEN LEU2 The FAPoptim-MYC-Linker sequence was cloned into the BamHI and SmaI sites of the pRS415-TEF1pr plasmid and the MFA1 signal sequence was cloned into the XbaI/SpeI sites upstream of the FAP tag to help target constructs to the ER.

pRS413-TEF1pr-MFA-NFAPoptim
TEF1pr-MFA1 signal sequence-NFAPoptim CEN HIS3 The FAPoptim-MYC-Linker sequence was cloned into the BamHI and SmaI sites of the pRS413-TEF1pr plasmid and the MFA1 signal sequence was cloned into the XbaI/SpeI sites upstream of the FAP tag to help target constructs to the ER.

pRS415-MET25pr
MET25pr CEN LEU2 The TEF1 promoter from the pRS415-TEF1pr plasmid (Mumberg et al., 1995) was removed by restriction digestion and replaced with the MET25 promoter (PCR amplified from the pUG vector series) flanked by SacI and XbaI restriction sites.

MET25pr CEN LEU2
The FAPoptim-MYC-Linker sequence was cloned into the BamHI and SmaI sites of the pRS415-MET25pr plasmid.

MET25pr CEN LEU2
The Linker-MYC-FAPoptim-MYC sequence was cloned into the SalI and XhoI sites of the pRS415-MET25pr plasmid.

pRS413-MET25pr
MET25pr CEN HIS3 The TEF1 promoter from the pRS413-TEF1pr plasmid (Mumberg et al., 1995) was removed by restriction digestion and replaced with the MET25 promoter (PCR amplified from the pUG vector series) flanked by SacI and XbaI restriction sites.

MET25pr CEN HIS3
The FAPoptim-MYC-Linker sequence was cloned into the BamHI and SmaI sites of the pRS413-MET25pr plasmid.

MET25pr CEN HIS3
The Linker-MYC-FAPoptim-MYC sequence was cloned into the SalI and XhoI sites of the pRS413-MET25pr plasmid.

pRS415-CUP1pr
CUP1pr CEN LEU2 The TEF1 promoter from the pRS415-TEF1pr plasmid (Mumberg et al., 1995) was removed by restriction digestion and replaced with the CUP1 promoter (PCR amplified from the pKK212 plasmid used in (O'Donnell et al., 2013) flanked by SacI and BamHI restriction sites.

pRS413-CUP1pr
CUP1pr CEN HIS3 The TEF1 promoter from the pRS413-TEF1pr plasmid (Mumberg et al., 1995) was removed by restriction digestion and replaced with the CUP1 promoter (PCR amplified from the pKK212 plasmid used in (O'Donnell et al., 2013) flanked by SacI and BamHI restriction sites.

Genotype
Reference or Description Addgene ID pRS413-TEF1pr-SEC61-NFAP TEF1pr-SEC61-NFAP CEN HIS3 The coding sequence of SEC61 lacking the stop codon was PCR amplified with primers containing BamHI and SmaI restriction enzyme adaptors.The PCR product was then inserted into the pRS413-TEF1pr-NFAPoptim plasmid using standard cloning approaches.

Figure
data quantified by flow cytometry in panel 3C of the main paper, with FAPOPTIM-Ste3 in magenta and CMAC in blue.(F) Histograms of flow cytometry data showing fluorescence distributions for cells exposed to the pHs indicated and expressing FAPOPTIM-Ste3 and stained with either MG-ESTER (top) or MG-TAU (bottom).(G) The percentage of cells remaining in each experiment performed for Figure 3D-E after the non-fluorescent cells were removed by gating.While no significant pH-dependent changes were observed for MG-ESTER-FAPOPTIM-Ste3 in the population of fluorescent cells (top), there was a dramatic loss in fluorescence for the MG-TAU incubated cells (bottom).Student's t-tests were used to compare FAPOPTIM-Ste3 cells imaged at lower pH to those imaged at pH 7.0 (not significant = ns; p < 0.05 = *).Supplemental Figure S3 (accompanies Figure 4): Inducing FAP expression from MET25 or CUP1 promoters.(A & D) Confocal fluorescence microscopy of soluble FAPOPTIM (magenta) expressed in WT cells from plasmids containing the Met25pr (A) or Cup1pr (D), with CMAC (blue) marking vacuoles.Images were captured at the times indicated and the empty vector control shows the background fluorescence when no FAP is expressed from these promoters.(B & E) Mean whole cell fluorescence intensity of FAPOPTIM signal from confocal microscopy in panels A and D, respectively.Kruskal-Wallis with Dunn's post hoc tests was performed, and statistical comparisons were made relative to the t=0 control.(p<0.05= *; p<0.005 = **; p<0.0005 = ***).(C & F) Immunoblot of whole cell extracts from WT cells expressing the FAPOPTIM from the indicated promoter over time.MW markers are indicated on the left in kilodaltons.Supplemental Figure S4 (accompanies Figure 4): FAP-expression and -tagging plasmids.(A) Map of N-terminal FAP tagging plasmid with ER-targeting sequence from Mfa1 added to allow ER insertion.(B) Map of C-terminal FAP integration cassette marked with KANMX4.
were incubated with MG-TAU (impermeant) dye at t=0 to visualize cell surface Ste3 (magenta) and CMAC (blue) stained the vacuoles.The dye was then washed from the cells before imaging at the indicated times, allowing us to monitor the steady-state turnover of FAP-Ste3 from the PM.(B) Kruskal-Wallis statistical analysis with Dunn's post hoc test was performed to compare the means of the three replicates to t=0 control for each of the strains (not significant = ns; p < 0.05 = *; p < 0.005 = **;p <0.0005 = ***).PM. (A) Cells expressing FAPOPTIM-Ste3 from a plasmid using the STE3pr and chromosomally integrated Ste3-pHluorin (green) were imaged by confocal fluorescence microscopy.Cells were incubated with MG-ESTER (magenta) and CMAC dye (blue).Once the MG-ESTER dye was washed from the cells, 5 µM of a-factor was added, and cells were imaged at 60 min with no further dye additions.This allowed us to monitor a single pool of Ste3 at the onset of the experiment with FAP while monitoring the total pool of Ste3 with pHluorin.These images are added in support of the data shown in Figure 11C.(B) Line scan analysis to monitor the relative distributions of FAP-Ste3 and Ste3-pHluorin after pheromone addition.The region used in the line scan is indicated as a dashed yellow line on the image and the fluorescence intensities along this line for GFP (left y-axis) or FAP (right y-axis) are plotted.In each case, the Ste3-pHluorin is distributed to the bud tip while the FAPOPTIM-Ste3 localizes to the regions adjacent to the shmoo tip.
The pRS415-TEF1pr plasmid(Mumberg et al., 1995) was used to insert the Ste3 coding sequence fused at the Cterminus to GFP.The GFP was inserted into the plasmid first between the XhoI and SalI restriction enzyme sites and then Ste3 coding sequence (lacking the stop codon) was inserted into the SpeI and SmaI restriction enzyme sites.The pRS415-TEF1pr-MFA-NFAPoptim plasmid (described above) was used to insert the Ste3 coding sequence as an in-frame N-terminal fusion to FAP.The Ste3 coding sequence (lacking the stop codon) was inserted into the SmaI and XhoI restriction enzyme sites.The resulting plasmid contains the MFA1 ER targeting and secretion sequence, an Nterminal FAPoptim tag on Ste3 the STE3 coding sequence lacking amino acids 288-end (the C-tail of Ste3), where amino acid 288 was converted to a stop codon via PCR, was preformed and this DNA was subcloned into pRS415-STE3pr-MFA-Igkappa-FAP plasmid at the SmaI and XhoI sitesposition 424 in Ste3's Ctail was mutated to arginine using sitedirected mutagenesis of pRS415-STE3pr-MFA-Igkappa-FAP-STE3. of ANP1 lacking the stop codon was PCR amplified with primers containing BamHI and SmaI restriction enzyme adaptors.The PCR product was then inserted into the pRS413of ERG6 lacking the stop codon was PCR amplified with primers containing BamHI and SmaI restriction enzyme adaptors.The PCR product was then inserted into the pRS413-TEF1pr-NFAPoptim plasmid using standard cloning approaches.221121pRS413-TEF1pr-PMA1-NFAPTEF1pr-PMA1-NFAP CEN HIS3The coding sequence of PMA1 lacking the stop codon was PCR amplified with primers containing BamHI and SmaI restriction enzyme adaptors.The PCR product was then inserted into the pRS413of RPA34 lacking the stop codon was PCR amplified with primers containing BamHI and SmaI restriction enzyme adaptors.The PCR product was then inserted into the pRS413of SEC7 lacking the stop codon was PCR amplified with primers containing BamHI and SmaI restriction enzyme adaptors.The PCR product was then inserted into the pRS413-TEF1pr-NFAPoptim plasmid using standard cloning approaches.